PHOTOCHEMICAL REGULATION OF CALCIUM IN CELL PHYSIOLOGY

细胞内Ca 2+浓度的变化控制着无数的 包括肌肉收缩、分泌、有丝分裂 通道门控、趋化性和气孔关闭。 在过去 十年来，两种技术彻底改变了钙离子的研究 参与细胞生理学：使用 比率式Ca 2+特异性荧光指示剂，如fura-2;以及 细胞底物如ATP，cGMP， 等从生物惰性或“笼”的形式通过闪光光解 技术. 这项建议的目的是利用快速光化学 控制二价阳离子水平以表征机制， 调节细胞生理过程。 Ca 2+特异性 光不稳定螯合剂将被开发并用于操纵 细胞内Ca 2+浓度独立于其他效应物，如 如Mg 2，ATP，GTP等，因此这些物种的调节作用 对Ca 2+依赖性细胞生理学的影响。 此外，A 将合成Mg 2+特异性光不稳定螯合剂。 由于Mg 2+是 在核苷酸依赖性过程中的一个必要的辅因子，快速 阳离子的光化学释放将用于引发这些 处理和表征Mg 2 +/核苷酸复合物的作用， 例如，肌肉收缩和分泌的机制。 这些 研究将扩展那些已经用DM-硝基酚， 基于EDTA的光不稳定螯合剂，由申请人开发， 以高亲和力结合Ca 2+和Mg 2+。 这一建议的基础是 是一种新的钙离子特异性光不稳定螯合剂， 这是最近合成的。 将使用这种笼状Ca 2+， 结合荧光Ca 2+指示剂，研究其动力学， 调节黑素营养细胞的分泌事件和 心脏和平滑肌，因为Ca 2+是关键的细胞内第二 这些系统中的信使。 其中许多进程分布在 病理状态。 在充分描述这些疾病之前 状态可以给出，非疾病状态的更完整的理解 应该完成。 拟议的研究将有助于 更好地了解这些过程的正常运作。

Changes in intracellular Ca2+ concentration control a myriad of physiological processes including muscle contraction, secretion, mitosis, channel gating, chemotaxis and stomatal pore closure. During the past ten years two techniques have revolutionized the study of Ca2+ involvement in cell physiology: measurement of Ca2+ concentrations using ratiometric, Ca2+ specific fluorescent indicators such as fura-2; and the rapid increase in concentration of cellular substrates such as ATP, cGMP, etc. from a biologically inert or 'caged' form by flash-photolysis techniques. The objective of this proposal is to use rapid photochemical control of divalent cation levels to characterize the mechanism and regulation of cellular physiological processes. Ca2+-specific photolabile chelators will be developed and used to manipulate intracellular Ca2+ concentrations independently of other effectors, such as Mg2, ATP, GTP, etc. so that the regulatory roles that these species have on Ca2+-dependent cell physiology can be defined. Additionally, a Mg2+-specific photolabile chelator will be synthesized. Since Mg2+ is a necessary co-factor in nucleotide-dependent processes, the rapid photochemical release of the cation will be used to initiate these processes and characterize the role of Mg2+/nucleotide complexes in, for example, the mechanisms of muscle contraction and secretion. These studies will extend those already performed with DM-nitrophen, a photolabile chelator based on EDTA, developed by the applicant, which binds both Ca2+ and Mg2+ with high affinity. The basis of this proposal is a new Ca2+-specific photolabile chelator called nitrophenyl-EGTA, which has been recently synthesized. This caged Ca2+ will be used, in combination with fluorescent Ca2+ indicators, to study the kinetics and regulation of secretion events in melanotrophs and of contraction in cardiac, and smooth muscle because Ca2+ is the key intracellular second messenger in these systems. Many of these processes are distributed in pathological states. Before an adequate description of these disease states can be given, a more complete understanding of non-disease states should be accomplished. The proposed studies will contribute to a greater understanding of the normal functioning of these processes.